Synthesis of hpGRF (Somatocrinin) in liquid phase and intermediate peptides

ABSTRACT

The invention relates to synthesis of hpGRF (Somatocrinin) in liquid phase and to intermediate peptides, comprising:-coupling, one after the other and in the order of the sequence of the GRF, the fragments in which: (a) the side acid functions of the aspartic and glutamic acids and the side amine function of the lysine are protected by protector groups stable in the conditions of deprotection of the group Boc, (b) the guanidine function of the arginine is protected by protonation, and (c) the N-terminal amino acid is protected on the amine by the Boc group;-selectively eliminating the group Boc from the N-terminal amine of the peptide in phase of elongation by hydrolysis with trifluoroacetic acid, said coupling being effected in an aprotic polar solvent and-eliminating, at the end of sequence, all the protector groups by hydrolysis with the aid of a 0.1 to 1M solution of methanesulfonic or trifluoromethanesulfonic acid in trifluoroacetic acid.

The present invention relates to a synthesis of hpGRF (Somatocrinin) inliquid phase and intermediate peptides.

hpGRF (human Pancreatic Growth Hormone releasing Factor) or Somatocrininis a peptide constituted by the chain formation of 44 amino acids. Itssequence is as follows: ##STR1##

It has recently been discovered by A. GUILLEMIN et coll. (Science, 218,585-587 (1982) from extracts of a human pancreatic tumour.

This peptide is particularly active on the stimulation of the release ofthe growth hormone (GH) both in vitro and in vivo. In vitro, inparticular, its effectiveness is shown at doses of some fento moles/ml(ED₅₀ =15 fento moles/ml). The therapeutic interest of this substance inhuman medicine will therefore lie in the treatment of dwarfism andretarded growth in pediatrics. Other applications are possible in thecases of anabolic protein deficiency (stress-related ulcers, repair offractures or of wounds of the cartilage, extensive burns (during theanabolic phase), cutaneous repairs, osteoporoses).

In the veterinary domain, the interest of this compound in the weightgrowth of farm-breeding animals (beef-cattle, sheep, pigs, chicken, . .. ) and in the increase in lactation (cows, ewes) is obvious.

The industrial development of this polypeptide compound necessitates thesynthesis of large quantities of this substance. Conventional processesof synthesis in solid phase allow small quantities of this activeprinciple to be prepared in short periods of time (Science, 218, 585-587(1982) but at very high costs which are incompatible with a large-scalepharmaceutical development.

The present invention describes a process of synthesis in liquid phasewhich may be carried out on an industrial scale, allowing access to theactive principle with excellent yield and rate of purity. This processis based on the principle of synthesis by fragments.

The process of the present invention is characterized by the step ofcoupling, one after the other and in the order of the sequence, thefragments in which:

(a) the side acid functions of the aspartic and glutamic acids and theside amine function of the lysine are protected by protector groupsstable in the conditions of deprotection of the Boc group(tertiobutoxycarbonyl),

(b) the guanidine function of the arginine is protected by protonation,and

(c) the N-terminal amino acid is protected on the amine by the Bocgroup,

(d) by selectively eliminating the Boc group from the N-terminal amineof the peptide in elongation phase by hydrolysis with trifluoroaceticacid, said coupling being effected in an aprotic polar solvent, and atthe end of sequence all the protector groups are eliminated byhydrolysis with the aid of a 0.1 to 1M solution of methanesulfonic ortrifluoromethanesulfonic acid in trifluoroacetic acid.

The process of the present invention may also be applied to thesynthesis of hpGRF-1-40, a natural product also isolated by R.GUILLEMIN, which is almost as active as the hpGRF-1-44 and able to beused in the same therapeutic applications.

The process of synthesis of the GRF is further characterized by thefollowing features:

Application of the principle of minimum protection to the functionalizedside chains.

Temporary protection of the side guanidine function of the arginine bythe nitro group. The arginine is introduced in sequence in the nitroguanidine form. The nitro function is then eliminated as soon aspossible by catalytic hydrogenation with the aid of Pd/charcoal, or byusing in place of gaseous hydrogen a generator of hydrogen such asformic acid or ammonium formate. In this way, all the synthesizedfragments possessing arginine in their sequence have, at the end ofsynthesis, the guanidine functions simply protected by protonation withthe aid of a strong acid (hydrochloric acid, for example).

The carboxylic acid functions of the side chains of the glutamic andaspartic acid are protected by groups cleavable by catalytichydrogenation (H₂ /Pd/ charcoal) or in a strong acid medium such as themethanesulfonic acid (0.5 M)--trifluoroacetic acid, ortrifluoromethanesulfonic acid (0.5 M)--trifluoroacetic acid mixtures.The invention recommends as protectors benzyl (O Bzl) or 2,6dichlorobenzyl ester. These protector groups are stable in theconditions of intermittent deprotection of the amines in alpha(elimination of the t-butyloxycarbonyl (Boc) groups by trifluoroaceticacid).

The amine functions of the side chains of the lysines are protected bygroups cleavable under the same conditions as previously. The inventionrecommends the benzyloxycarbonyl (Z), 2-chloro or 2-bromobenzyloxycarbonyl (2-Cl or 2-Br Z) groups stable in the conditions ofintermittent deprotection by trifluoroacetic acid.

The hydroxyl functions present in the threonine, serine and tyrosine arenot protected.

The elongation of the peptide from the synthesized fragments is effectedby using as coupling agent the hexafluorophosphate of benzotriazolyloxyphosphonium (BOP), or dicyclohexyl carbodiimide in the presence of1-hydroxy benzotriazole, or according to the method employingcarboxyazides (Curtius), in an appropriate solvent such as dimethylformamide or dimethylsulfoxide. Isolation of the product from thereaction medium is effected by introducing a third solvent which rendersinsoluble (ether, ethyl acetate, . . . ) which precipitates the peptide.

One is limited during the steps of coupling to summary purifications ofthe solid-phase washing type with the aid of appropriate solvents inorder to eliminate the slight excess of the last coupled fragment, aswell as the impurities brought by the coupling agents.

Each fragment coupling operation is followed by a phase of intermittentdeprotection of the Boc tertiobutoxycarbonyl protector group, at thelevel of the amine on which the following coupling will be effected.Such deprotection is ensured by trifluoroacetic acid in methylenechloride (50/50 by volume).

The deprotections of the side chains at the end of elongation of thepeptide may be effected by hydrogenation in the presence of a catalyst(such as Pd/C) with the aid of gaseous hydrogen under a slight pressure(1 to 5 kg) or with a generator of hydrogen such as formic acid orammonium formate. It is also possible to eliminate this type ofprotector group by a strong acid such as the mixtures of methanesulfonicacid in trifluoroacetic acid (0.5M) or of trifluoromethanesulfonic acidin trifluoroacetic acid (0.5M).

The product, after the terminal deprotections, is purified by filtrationover Sephadex gel G 50 with the acid of 30% acetic acid. The GRF-1-44enriched fractions are gathered together and subjected to achromatography on ion exchangers (cations) of the carboxy type and withthe aid of a gradient with increasing ionic force adapted to the type ofion exchanger resin used. The fractions of highest purity are gatheredtogether and purified, either by partition chromatography on anappropriate support of the Sephadex G 50 or Biogel P 10 type, or bycounter-current distribution.

The fractions of which the titer of purity is judged satisfactory byanalytic HPLC are gathered together. The others are recycled.

A variant of the process consists in replacing the partitionchromatography or counter-current distribution by preparative HPLC.

The fragments used for the synthesis of hpGRF-1-44 are determined on thebasis of chemical considerations taking particular account of theminimum risks of racemization during the fragment coupling phases.

Each of the fragments is then synthesized according to a strategyadapted to each case in accordance with a step-wise process in liquidphase.

Scheme 1 hereinafter represents one of the strategies of synthesis whichmay be used for the synthesis of hpGRF-1-44.

The vertical arrows delimit the fragments used in the synthesis and eachof them is designated by a letter:

A: fragment 40-44

B: fragment 33-39

C: fragment 28-32

D: fragment 25-27

E: fragment 21-24

F: fragment 16-20

G: fragment 12-15

H: fragment 5-11

I: fragment 1-4

In addition, the protections of the side chains have been symbolized by:

X₁ =ester of benzyl type (O Bzl)

X₂ =carbamate protection of the benzyloxycarbonyl (Z) type.

Tables I to IX thereafter indicate the process of synthesis used foreach of fragments A to I. ##STR2##

                                      TABLE I                                     __________________________________________________________________________    (fragment A)                                                                  __________________________________________________________________________     ##STR3##                                                                     __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    (fragment B)                                                                  __________________________________________________________________________     ##STR4##                                                                      ##STR5##                                                                     __________________________________________________________________________

                  TABLE III                                                       ______________________________________                                        (fragment C)                                                                  ______________________________________                                         ##STR6##                                                                     ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        (fragment D)                                                                  ______________________________________                                         ##STR7##                                                                     ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        (fragment E)                                                                  ______________________________________                                         ##STR8##                                                                     ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        (fragment F)                                                                  ______________________________________                                         ##STR9##                                                                      ##STR10##                                                                    ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        (fragment G)                                                                  ______________________________________                                         ##STR11##                                                                     ##STR12##                                                                    ______________________________________                                    

                                      TABLE VIII                                  __________________________________________________________________________    fragment H                                                                    __________________________________________________________________________     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                    __________________________________________________________________________

    ______________________________________                                        (fragment I)                                                                  ______________________________________                                         ##STR16##                                                                    The hpGRF-1-44 may also be prepared in accordance with a process          

20-44: Peptide K

5-19: Peptide J

1-4: Peptide I

which are coupled in the following order:

5-19+20-44: 5-44

1-4+5+44: 1-44

Peptide J and peptide K are in turn synthesized by a process byfragments.

Peptide 5-19 is synthesized from 3 fragments and peptide 20-44 from 7fragments.

Schemes II and III hereinafter represent the strategies used forpreparing peptides J and K, peptide I having already been prepared inscheme I.

Scheme IV represents the coupling of the 3 peptides I, J and K to arriveat the hpGRF 1-44.

Tables X to XV show the schemes of synthesis of the fragments notdescribed on following scheme I.

Finally, in all cases, to obtain hpGRF 1-40, it suffices to reducefragment A to alaninamide. ##STR17##

                  TABLE X                                                         ______________________________________                                        (sub-fragment B.sub.1)                                                        ______________________________________                                        ______________________________________                                    

                  TABLE XI                                                        ______________________________________                                        (sub-fragment B.sub.2)                                                        ______________________________________                                         ##STR18##                                                                    ______________________________________                                    

                  TABLE XII                                                       ______________________________________                                        (fragment E.sub.1)                                                            ______________________________________                                         ##STR19##                                                                    ______________________________________                                    

                  TABLE XIII                                                      ______________________________________                                         (fragment F.sub.1)                                                           ______________________________________                                         ##STR20##                                                                    ______________________________________                                    

                                      TABLE XIV                                   __________________________________________________________________________     (fragment G.sub.1)                                                           __________________________________________________________________________     ##STR21##                                                                     ##STR22##                                                                    __________________________________________________________________________

                                      TABLE XV                                    __________________________________________________________________________     (fragment H.sub.1)                                                           __________________________________________________________________________     ##STR23##                                                                     ##STR24##                                                                    __________________________________________________________________________

The following examples will enable the scope of the invention to be morereadily understood.

The following abbreviations will be used.

The amino acids are represented by the symbols recommended by theNomenclature Commission of the IUPAC-IUB, Biochemistry Section.

Ala: Alanine

Arg: Arginine

Asn: Asparagine

Asp: Aspartic acid

Gln: Glutamine

Glu: Glutamic acid

Gly: Glycine

Ile: Isoleucine

Leu: Leucine

Lys: Lysine

Met: Methionine

Phe: Phenyl alanine

Ser: Serine

Thr: Threonine

Tyr: Tyrosine

Val: Valine

With the exception of glycine, they all have the L-configuration.

    __________________________________________________________________________    CCM     thin layer chromatography                                             OBzl    benzylic ester                                                                                      ##STR25##                                       Z       benzyl oxycarbonyl (carbamate)                                                                      ##STR26##                                       Boc     tertiobutoxycarbonyl (carbamate)                                                                    ##STR27##                                       DMF     dimethyl formamide                                                    NEM     Nethyl morpholine                                                     TFA     trifluoroacetic acid                                                  MA      method of coupling with mixed anhydrides                              ONSU    ester activated with Nhydroxy succinimide                                                           ##STR28##                                       ONp     ester activated with ortho nitro phenol                                                             ##STR29##                                       Troc    trichloro ethoxy carbonyl (carbamate)                                                               ##STR30##                                       OBu.sup.t                                                                             ester with tertiobutanol                                                                            ##STR31##                                       OTcp    ester activated with 2,3,5                                                    trichlorophenol                                                       OHBT    Nhydroxy benzotriazole                                                ONb     ester activated with Nhydroxy 5-norbornene 2,3-dicarboximide                                        ##STR32##                                       BOP     hexafluorophosphate of benzo triazolyl oxyphosphonium                                               ##STR33##                                       DCHa    dicyclohexylamine                                                     DCU     dicyclohexylurea                                                      DCC or DCCl                                                                           dicyclohexylcarbodiimide                                              TA      ambient temperature                                                   DIPEA   diisopropyl ethyl amine                                               EPP     polypeptidic purity                                                   TFMSA   trifluoro methane sulfonic acid                                       AAA     analysis of amino acids                                               HPLC    high performance liquid chromatography                                __________________________________________________________________________

Media of chromatography expressed in volumes:

    ______________________________________                                        BEW.sub.1 butanol, AcOH, H.sub.2 O 72/7/21                                    BEW.sub.2 butanol, AcOH, H.sub.2 O 67/10/23                                   BPEW.sub.1                                                                              butanol, pyridine, AcOH, H.sub.2 O 50/12/12/25                      EPAW      AcOEt, pyridine, HCO.sub.2 H, H.sub.2 O 63/21/10/6                  BPEW.sub.2                                                                              butanol, pyridine, AcOH, H.sub.2 O 42/24/4/30                       ______________________________________                                    

EXAMPLE I Synthesis of H-Ala-Arg-Ala-Arg-Leu-NH₂ (fragment A) ##STR34##

130 g of leucine amide (H-Leu-NH₂) are dissolved at ambient temperaturein 1.5 l of DMF. 333 g of Boc-Arg(NO₂)-OH then 500 g of BOP are added.The pH is adjusted to 7 with pH paper (on small samples diluted withwater) and with the aid of N-ethylmorpholine (NEM). The medium isstirred and the development of the reaction is followed by TLC. Thereaction is terminated after 4 hours. The medium is evaporated todryness in vacuo at 25°. The residue is taken up in 1 liter of water andsolid is obtained which is washed with water, then with a 5% aqueoussolution of NaHCO₃ with water, with ethyl acetate and, finally, thesolid is dried in air. It is monitored by TLC.

The preceding solid is introduced into 2 l of a 50-50 by volume mixtureof trifluoroacetic acid/methylene chloride. The medium is stirred for 10mins. at ambient temperature and evaporated to dryness in vacuo atambient temperature. The residue of evaporation is taken up in ether,drained, dried and monitored by TLC and NMR.

Yield: 339 g (90%) expressed in trifluoroacetate of a white solid.

2. H-Ala-Arg(NO₂)Leu-NH₂

443 g of trifluoroacetate of H-Arg(NO₂)Leu-NH₂ are dissolved in 2 l ofDMF. 200 g of Boc-Ala-OH, then 500 g of BOP are added. The pH isadjusted to 7 with pH paper (on small samples of the reaction medium)with the aid of NEM. The medium is stirred and the development of thereaction is followed by TLC. The reaction is terminated after 4 hrs. Themedium is evaporated to dryness in vacuo at 25°. The residue is taken upin 2 l of water and 2 l of ethyl acetate. The organic phase is washedwith a 5% aqueous solution of NaHCO₃, with water, dried and evaporated.The tripeptide is recrystallized in ethyl-acetate/ether, and finallydried in vacuo. It is monitored by TLC and NMR.

The preceding product, dried, is treated with a 50-50 (by volume)mixture of TFA-CH₂ -Cl₂ under the conditions of the preceding ExampleI-1). Isolation is also effected under the same conditions.

Yield: 412 g (80%) expressed in trifluoroacetate of a white pulverulentproduct (monitored in TLC and NMR).

3. Arg(NO₂)-Ala-Arg(NO₂)-Leu-NH₂

From 515 g of H-Ala-Arg(NO₂)-Leu-NH₂ in 2.5 l of DMF and 333 g ofBoc-Arg(NO₂)-OH and 500 g of BOP, 607 g (85%) of a white solid monitoredby TLC and NMR are obtained by employing the operational conditionsdescribed in Example I-1, after treatment with the mixture TFA-CH₂ Cl₂.

4. Z-Ala-Arg(NO₂)-Ala-Arg-Leu-NH₂

From 715 g of trifluoroacetate of E-Arg(NO₂)Ala-Arg(NO₂)-Leu-NH₂ insolution in 4 l of DMF and 233 g of Z-Ala-OH and 500 g of BOP employingthe same technique as the one described in I-2 (and without treatment inthat case by TFA-CH₂ Cl₂), 612 g of Z-Ala-Arg(NO₂)-Ala-Arg(NO₂)Leu-NH₂(76%) are obtained, after recrystallization in the DMF-ether mixture, inthe form of a white pulverulent solid, monitored by TLC and NMR.

5. Ala-Arg-Ala-Arg-Leu-NH₂, 3 HCl

200 g of Z-Ala-Arg(NO₂)Ala-Arg(NO₂)-Leu-NH₂ (0.25 mole) are suspended in2 l of methanol containing 0.8 mole of HCl. 40 g of Pd/C with 10% of Pdare added, and the medium is stirred in an atmosphere of hydrogen undera pressure of 1.2 bars, for 24 hours. After this interval of time, theend of the reaction is monitored by TLC. The catalyst is eliminated byfiltration and the solvent is evaporated in vacuo at ambienttemperature.

The solid residue is purified by chromatography over silica gel, usingan elution medium the (50-12-12-25 by volume) mixture of butanol,pyridine, HO₂ CCH₃, OH₂.

The fractions containing the pure product are collected together,evaporated and lyophilized.

Yield: 119 g (69%) of a white pulverulent solid

Monitored by: NMR, TLC.

Analysis of aminoacids: Leu 1.03 (1), Arg 1.92 (2), Ala 1.95 (2).

EXAMPLE II Synthesis of Boc-Glu(OBzl)-Arg-Gly-OH, HCl (fragment B₁) 1.Z-Arg(NO₂)-Gly-OBzl

3.37 g (0.01 mole) of tosylate of H-Gly-OBzl are dissolved in 15 ml ofDMF, then 1 equivalent of NEM (1.3 ml) is added. This solution is addedto a solution of 3.53 g (0.01 mole) of Z-Arg(NO₂)-OH in 15 ml of DMFcontaining 1 equivalent of NEM (1.3 ml). 4.5 g (0.01 mole) of BOP arethen added to the reaction mixture whose pH is then brought to 7 byaddition of NEM and which is stirred at ambient temperature for 2 hours30 minutes (the end of the reaction is determined by TLC:chloroform-methanol (3/1).

The reaction mixture is evaporated to dryness under reduced pressure(0.1 mm of mercury) at a temperature lower than 30° C. The residue istaken up in 100 ml of ethyl acetate. This solution is poured into 120 mlof strongly stirred iced water. After a few instants of stirring, aprecipitate is formed which is left overnight in the refrigerator.

The solid is drained and washed successively in the solid state with:

2×100 ml of an aqueous solution of sodium bicarbonate

2×100 ml of a 5% aqueous solution of SO₄ HK-SO₄ K₂

2×100 ml of water

2×100 ml of ether

then dried in vacuo (0.1 mm of mercury) up to a constant weight.

Yield: 4 g (80%) Koffler melting point: 149° C. TLC: chloroform-methanol(3/1) Rf: 0.73 Monitored by NMR.

2. HCl-H-Arg-Gly-OH

100 g (0.2 mole) of Z-Arg(NO₂)-Gly-OBzl are suspended in a mixture of600 ml of water, 600 ml of N hydrochloric acid and 100 ml oftetrahydrofuran. 60 g of 10% Pd/C containing 50% humidity are added.This mixture is hydrogenated for 48 hours at ambient temperature andunder a pressure of 15 mm of mercury. The catalyst is then filtered andthe aqueous solution is taken to pH 6.5 by addition of amberlite IR 45resin (OH form). The resin is drained. The solution is then taken to pH8.5 by addition of a fresh quantity of resin and the whole is stirredfor 20 minutes in a rotavapor in vacuo (25 mm of mercury). The resin isdrained and the absence of ammonium chloride in the aqueous solution ischecked by the Nesler test, then this solution is evaporated to drynessunder reduced pressure (0.1 mm of mercury) at a temperature lower than30° C. A gummy residue is obtained which is dried overnight in adesiccator with phosphoric anhydride.

Yield: 43.19 g (80.6%) TLC: ethyl acetate-pyridine-formic acid-water(40-21-10-6) Rf: 0.1 Sakaguchi test positive (red spot) Monitored byNMR.

3. Boc-Glu(OBzl)-Arg(HCl)-Gly-OH

41.5 g of HCl-H-Arg-Gly-OH (0.155 mole) are dissolved in a mixture of400 ml of DMF and of 130 ml of water. The pH is adjusted to 7 byaddition of N hydrochloric acid.

To this solution are simultaneously added:

a solution of 68.8 g of Boc-Glu(OBzl)-ONP (0.150 ml) in a mixture of 100ml of DMF and 40 ml of water.

a solution of 22.7 g of hydroxybenzotriazole (0.150 mole) in a mixtureof 100 ml of DMF and 40 ml of water.

a solution of 37.8 ml of NEM (2×0.150 mole) in 100 ml of DMF.

The reaction mixture is stirred at ambient temperature and the reactionis followed in TLC (chloroform-methanol-acetic acid 95-5-9 andpyridine-ethyl acetate-formic acid-water 21-40-10-6).

After one hour of reaction, 13.76 g of Boc-Glu(OBzl)-ONP are added.

After 2 hours of reaction, 13.76 g of Boc-Glu-OBzl)-ONP are added.

After 3 hours of reaction, 7 g of Boc-Glu(OBzl)-ONP are added and thereaction is continued for 1 hour.

The reaction mixture is then evaporated to dryness under reducedpressure (0.1 mm of mercury) and at a temperature lower than 30° C. Athick oil is obtained which is dissolved in 500 ml of ethyl acetate. Byaddition of 1 liter of ether, a thick oil is formed. It is left to stand24 hours in the refrigerator, then the liquid phase is decanted.

The residual oil is taken up in 500 ml of methanol. To this solution areadded 500 g of silica (70-230 mesh) and the solvent is evaporated bymeans of a rotavapor. The powder obtained is mixed with a 80-20 mixtureof chloroform-methanol and the gel formed is introduced at the top of acolumn of silica gel (height 200 cm, diameter 85 mm) mounted in the80-20 chloroform-methanol mixture.

The product is eluted with:

80-20 mixture of chloroform-methanol: 25 liters.

50-50 mixture of chloroform-methanol: 10 liters

methanol: 30 liters.

The purification is followed by TLC (pyridine-ethyl acetate-formicacid-water, 21-40-10-6)

2 fractions are collected:

fraction A, 29.80 g

TLC: pyridine-ethyl acetate-formic acid-water, 21-40-10-6; Rf: 0.75

Monitored by NMR

HPLC--EPP: 96.98%

AAA: Glu 1.01-Gly 1.01-Arg 0.98

Fraction B, 36.82 g

TLC: pyridine-ethyl acetate-formic acid-water, 21-40-10-6; Rf: 0.75

1H NMR spectrum conformable

HPLC--EPP: 99.61%

AAA: Glu 0.93-Gly 0.99-Arg 1.08

EXAMPLE III Synthesis of Boc-Glu(OBzl)-Ser-Asn-Gln-OH (fragment B2) 1.Boc-Asn-Gln-OBzl

TFA, H-Gln-OBzl (0.445 mole) are dissolved in 500 ml of DMF. To thissolution are successively added:

103.3 g of Boc-Asn-OH (0.445 mole)

62.3 ml of NEM

217.8 g of BOP (0.5 mole)

then the pH of the solution is returned to 7 by addition of NEM.

The mixture is stirred at ambient temperature, the pH is maintained at 7by addition of NEM if necessary. The reaction is followed in TLC (3-1chloroform-methanol). After one hour, the reaction is terminated and theproduct is precipitated by addition of 1.5 liters of ethyl acetate. Itis left one hour with stirring then left overnight in the refrigerator.

The solid is drained, washed with ethyl acetate (3×500 ml), with ether(1 liter), then dried.

Yield: 108.13 g (53.9%),

TLC: 3-1 chloroform-methanol; Rf: 0.63.

2. TFA, H-Asn-Gln-OBzl

102 g of Boc-Asn-Gln-OBzl (0.226 mole) are dissolved in 500 ml of TFAcooled in a bath of ice. The bath of ice is removed and stirring iscontinued at ambient temperature for 15 minutes. A light insolublesubstance is drained and stirring is continued for 15 minutes. Thereaction medium is concentrated to 1/4 under reduced pressure (25 mm ofmercury) and is taken up in 300 ml of ether. This operation is repeatedtwice (2×300 ml of ether) and the remaining solvent is eliminated invacuo (25 mm of mercury).

A hygroscopic white solid is obtained.

Yield: 107 g, TLC: 3-1 chloroform-methanol; Rf: 0.08.

3. Boc-Ser-Asn-Gln-OBzl

0.226 mole of TFA, H-Asn-Gln-OBzl is dissolved in 1.5 liters of DMF. Tothis solution are successively added:

48.4 g of Boc-Ser-OH, 1/2 H₂ O (0.226 mole)

34.8 ml of NEM (0.27 mole)

111 g of BOP (0.25 mole)

then the pH of the solution is returned to 7 by addition of NEM.

The mixture is stirred at ambient temperature, maintaining the pH at 7by addition of NEM if necessary. The development of the reaction isfollowed in TLC (chloroform-methanol-acetic acid, 9-2-0.5). After 2hours, the reaction is terminated. A light insoluble substance isfiltered and the reaction medium is evaporated to dryness under reducedpressure (0.1 mm of mercury) at a temperature lower than or equal to 30°C.

The residual oil is taken up in 800 ml of ethyl acetate. It is left for1 hour with stirring then left overnight to stand in a refrigerator.

The solid is drained, washed with ethyl acetate (2×200 ml) then withether (200 ml) and dried in vacuo.

Yield: 108.32 g (89%), TLC: chloroform-methanol-acetic acid, 9-2-0.5;Rf: 0.42.

4. TFA, H-Ser-Asn-Gln-OBzl

108 g of Boc-Ser-Asn-Gln-OBzl (0.2 mole) are dissolved in 520 ml of TFAcooled in an ice bath. The ice bath is removed and the product isstirred for 30 minutes at ambient temperature. A light insolublesubstance is filtered, then evaporated to dryness under reduced pressureat a temperature lower than 30° C. The residue is taken up in ether, thesolid is drained, washed several times with ether and dried.

Yield: 139° C., TLC: tetrahydrofuran-pyridine-formic acid-water,60-20-10-6; Rf: 0.81.

5. TFA, H-Ser-Asn-Gln-OH

0.2 mole of TFA, H-Ser-Asn-Gln-OBzl are dissolved in 1 liter of water.22.4 g of 10% Pd/C containing 50% humidity are added to the solution.The mixture is hydrogenated at ambient temperature and under a pressureof 15 mm of mercury for 24 hours with stirring. The catalyst is filteredthen evaporated to dryness under reduced pressure (0.1 mm of mercury)and at a temperature lower than 30° C. The residue is left to dryovernight in a desiccator with phosphoric anhydride.

Yield: 93.66 g, TLC: tetrahydrofuran-pyridine-formic acid-water,60-20-10-6; Rf: 0.45.

6. Boc-Glu(OBzl)-Ser-Asn-Gln-OH

0.2 mole of TFA H-Ser-Asn-Gln-OH are dissolved in a mixture of 100 ml ofwater and of 600 ml of DMF. The pH of the solution is taken to 7 byaddition of NEM and a solution of 87.11 g of Boc-Glu(OBzl)-ONP (0.19mole) in 600 ml of DMF then 29.5 g of HOBT (0.19 mole) is added. The pHof the mixture is returned to 7 by addition of NEM and it is stirred atambient temperature: after one hour of stirring, 2.7 g ofBoc-Glu(OBlz)-ONP are added.

The reaction is followed in TLC (tetrahydrofuran-pyridine-formicacid-water, 60-20-10-6 and chloroform-methanol-acetic acid, 95-5-3).

Half an hour after the last addition, a light insoluble substance isfiltered, then evaporated to dryness under reduced pressure (0.1 mm ofmercury) at a temperature lower than 30° C. The oil obtained is taken upin 800 ml of ethyl acetate, a slightly gelatinous precipitate is formed.It is drained and washed with ethyl acetate (2×400 ml), then with etherand dried.

Yield: 114.3 g (90%).

The product must be washed again with ethyl acetate (1.2 liters) withstirring for 4 hours at ambient temperature. The solid is drained,washed with ether, dried.

Yield: 95.85 g (71.9%), TLC: tetrahydrofuran-pyridine-formic acid-water,60-20-10-6, Rf: 0.62.

n butanol-pyridine-acetic acid-water, 50-12-12-25-Rf: 0.46

1 H NMR, conformable

HPLC-EPP 87.81

AAA: Asn(asp): 1.01-Ser: 0.89-Glu(gln): 2.05

Melting point (Koffler): 134° C. dec.

EXAMPLE IV Synthesis of Boc-Ser-Arg-Gln-Gln-Gly-OH, HCl (fragment C) 1.Z-Gln-Gly-OMe

A suspension, cooled on an ice bath, of 75.36 g of HCl, H-Gly-OMe (0.6M)in one liter of DMF has 168.2 g of Z-Gln-OH (0.6), 256.41 g of BOP(0.7M) and 165.4 ml of N-ethyl morpholine (1.3M) added thereto. After 20hours of reaction at ambient temperature, the solution is concentratedunder reduce pressure and the residue taken up with ethyl acetate. Theorganic solution is washed by:

a solution of sodium bicarbonate

a solution of sodium chloride

a solution of KHSO₄ /K₂ SO₄

a solution of sodium chloride

The organic solution is dried over MgSO₄ and filtered. After 20 hours ofrest, there is crystallization in each of the solutions. Afterfiltration, the fractions coming from a crystallization in an aqueousmedium are collected together and washed in water-saturated AcOEt toyield after filtration and drying in vacuo 7.78 g of product (m.p.158°-159° C.).

The fraction coming from crystallization in AcOEt is washed inwater-satured AcOEt to yield, after filtration and drying in vacuo, 6.18g of product (m.p.: 150°-154° C.).

The aqueous phase and the organic phase give an additionalcrystallization. These latter two products are collected together andwashed as before to yield 38.05 additional g of comparable quality.

Overall yield: 107.63 g--monitored by NMR and TLC

2. HCl, H-Gln-Gly-OMe

A solution of 61.8 g of Z-Gln-Gly-OMe (175.9 mM) in 704 ml of DMF, 880ml of methanol and to which are added, with cooling by an ice bath, 176ml of an N HCl solution, then 3.1 g of 10% Pd/C. After 3 hr. 30 mins. ofhydrogenation under excess pressure of 35 cm of mercury, the catalyst isfiltered over cellite and the solution is concentrated under reducedpressure. The residue is used directly in the following step.

3. Boc-Gln-Gln-Gly-OMe

A solution of the residue obtained in the preceding step (theoretically175.9 mM) in 500 ml of DMF, cooled on an ice bath, has 77.6 g ofBoc-Gln-ONp (211.1 mM), 26.4 g of hydroxy benzotriazole (211.1 mM) andN-ethylmorpholine added thereto until a pH of 7 is obtained. After 20hrs. of reaction at ambient temperature, the solution sets en masse. Byaddition of ethyl acetate, a precipitate is obtained which is drained,washed with AcOEt and dried in vacuo. 64.27 g of product are obtained,viz. a yield of 82%.

Monitored by NMR and TLC.

4. TFA, H-Gln-Gln-Gly-OMe

A suspension cooled on an ice bath of 98.83 g of Boc-Gln-Gln-Gly-OMe(221.8 mM) in 300 ml of dichloromethane has 400 ml of TFA added thereto.After 30 mins. of reaction on an ice bath and one hour at ambienttemperature, the solution is concentrated under reduced pressure up tohalf its initial volume and the residue is poured over stirred ether.After filtration, washing and drying in vacuo, 119 g of product areobtained.

5. Boc-Arg(NO₂)-Gln-Gln-Gly-OMe

A solution of the 119 g of the preceding product in 1190 ml of DMFcooled on an ice bath has 77.91 g of Boc-Arg(NO₂)-OH (244 mM), 97.51 gof BOP (266.2 mM) and N-ethylmorpholine added thereto until pH 7 isobtained. After 20 hrs. reaction at ambient temperature, the solution ispoured over 8 liters of ethyl acetate. The precipitate obtained isfiltered, washed with AcOEt then dried in vacuo. 153.7 g of product areobtained. Monitored by NMR and TLC.

6. TFA, H-Arg(NO₂)-Gln-Gln-Gly-OMe

A suspension cooled on an ice bath of 153.7 g ofBoc-Arg(NO₂)-Gln-Gln-Gly-OMe (theoretically 221.8 mM) in 600 ml ofdichloromethane has 750 ml of TFA added thereto. After one hour atambient temperature, 250 ml of TFA are further added and 30 mins. later,250 ml of TFA are added. After a further hour of reaction, the solutionis concentrated to one third of the initial volume and the residue ispoured over 3 liters of ether with stirring. The precipitate formed isdrained, washed with ether and dried in vacuo. 167 g of product areobtained.

7. Boc-Ser-Arg(NO₂)-Gln-Gln-Gly-OMe

A solution cooled on an ice bath of 167 g of TFA,H-Arg(NO₂)-Gln-Gln-Gly-OMe (theoretically 221.8 mM) in 1.5 liters of DMFhas added thereto 97.52 g of Boc-Ser-ONb (266.2 mM), 33.31 g ofhydroxybenzotriazole (266.2 mM) and N ethylmorpholine until a pH of 7 isobtained. After 3 hrs. of reaction, at ambient temperature, part of theDMF is evaporated and the residual solution is poured over ethyl acetatewith stirring. The precipitate is filtered, washed with ethyl acetateand dried in vacuo. 153.3 g of product are obtained, viz. a yield of94.2% over 4 steps. Monitored by NMR and TLC.

8. Boc-Ser-Arg-Gln-Gln-Gly-OMe, AcOH

A solution of 153.3 g of Boc-Ser-Arg(NO₂)-Gln-Gln-Gly-OMe (208.9 mM) inone liter of methanol, one liter of water and 500 ml of acetic acid ishydrogenated for 20 hours in the presence of 10 g of 10% Pd/C. Afterfiltration of the catalyst and concentration of the solution, theresidue taken up in water is lyophilized. 153 g of product are obtained,viz. a yield of 97.8%.

9. Boc-Ser-Arg-Gln-Gln-Gly-OH, HCl

A solution of 104.4 g of Boc-Ser-Arg-Gln-Gln-Gly-OMe (134.4 mM) in 2liters of DMF has one liter of water, then 27.88 g of NaOH (697 mM) in50 ml of water added thereto, with cooling by an ice bath. After 15mins. of reaction at temperature close to 15° C., the product isneutralized by an N hydrochloric acid solution until a pH of 6.5 isobtained. After evaporation, the residue is triturated in ethyl acetate.The precipitate formed is drained, washed with AcOEt and dried in vacuo,then with air. 132.7 g of product are obtained.

Purification: 109 g of Boc-Ser-Arg-Gln-Gln-Gly-OH, HCl are purified bycounter-current distribution in the n-butanol-methanol-water (4-1-5)mixture. After 700 transfers, the product is fractioned into three partswhich, after evaporation and lyophilization, give: one fraction of 27.5g and two fractions of 23.79 g and 16.90 g to be repurified.

Monitored by NMR and TLC-AAA: Ser: 0.91-Arg: =0.97-Gln(Glu): 2.05-Gly:1.01

EXAMPLE V Synthesis of Boc-Asp(OBzl)-Ile-Met-NH-NH₂ (fragment D) 1.Boc-Met-NH-NH Troc

4.31 g (10 mM) of Boc-Met-OH, DCHa in solution in 50 ml of ethyl acetateare treated in the presence of 20 ml of water by a saturated solution ofpotassium bisulfate up to pH=3, the aqueous phase is extracted severaltimes with ethyl acetate and the extracts dried over magnesium sulfate.To this solution are added 2.28 g (11 mM) of H₂ N-NH-Troc(Troc=2,2,2-trichloro ethoxycarbonyl, this reagent being prepared inaccordance with YAJIMA, Chem. Pharm. Bull. 1971, 19, 420).

After cooling in an ice bath, 2.37 g (11 mM) of 97% DCC in solution in10 ml of ethyl acetate are added. After one night during which theproduct returns progressively to ambient temperature, thedicyclohexylurea is filtered and dried (2.04 g) and the organic solutionis washed successively with the following aqueous solutions:

5% sulfate-bisulfate twice ClNa 2M 2X 5% sodium bicarbonate twice, ClNa3M twice and finally with water twice. After drying over magnesiumsulfate and concentration of the solvent to virtual dryness, hexane isadded until cloudiness begins and the product is kept at +4° C.overnight after which the precipitate formed is filtered, washed with a(4/1) mixture of hexane and ethyl acetate and dried in vacuo; 3.46 g(79%) are thus obtained: m.p.=92°-4° alpha D 25°=-32°-C=1, dioxane.

TLC in chloroform-methanol-AcOH 95/5/3--Rf=0.45

2. TFA, H-Met-NH-NH Troc

43.9 g (0.1M) of Boc-Met-NH-NH-Troc in solution in 200 ml ofdichloromethane and 20 ml of ethane dithiol are treated with an ice bathin a nitrogen atmosphere and with stirring by 200 ml of trifluoroaceticacid, the cold bath is removed and the product is left with stirring forone hour. The product is isolated by elimination of the volatilereagents firstly under 20 mm then 0.1 mm of pressure, the residual oilis taken up twice with 75 ml of isopropanol by evaporating in vacuo thenwashed twice by 75 ml of hexane by decantation after which it is driedin vacuo in the presence of potassium hydroxide overnight, after whichit begins to solidify and is used as such in the following operation.

3. Boc-Ile-Met-NH-NH-Troc

The above oil (about 0.1M) of TFA, H-Met-NH-NH Troc in solution in 6500ml of ethyl acetate and cooled in an ice bath is treated with 12.7 ml(0.1M) of N ethyl morpholine then 14.85 g (0.1M) of HOBt, 1H₂ O then by34.35 g (0.08M) of Boc-Ile-OSu followed by 12.7 ml (0.1M) of Nethylmorpholine (NEM). After 1/2 hour, the cold bath is removed andsubsequently NEM is added periodically so as to maintain the apparent pHtowards 7.

After 20 hrs., the reaction is complete and isolation is effected bysuccessive washings with the following aqueous solutions: 5%sulfate-potassium bisulfate 3 times, water: three times, 5% sodiumbicarbonate: 3 times and finally, water, up to neutrality.

After drying over magnesium sulfate and evaporation of the solvent invacuo, 64 g of gum are obtained, chromatographed over a column of silicawith dichloromethane containing from 0 to 1.5% of methanol. 30 g (68%)of product having an HPLC purity of 98.9% and an NMR spectrum inaccordance with the expected structure, are thus obtained, m.p.:88°-92°.

4. TFA, H-Ile-Met-NH-NH-Troc

27.6 g (50 mM) of Boc-Ile-Met-NH-NH-Troc in solution in 140 ml ofdichloromethane and 14 ml of ethane dithiol are cooled with an ice bathand stirred in an atmosphere of nitrogen then 140 ml of trifluoroaceticacid are added thereto in 5-6 mins. The cold bath is removed and, after45 mins., the product is isolated by evaporation of the reagents to amaximum, take-up of the residual oil by 2×60 ml of isopropanol followedby evaporation and finally by 2 washings with 60 ml of pentane. Afterdrying for one night over potassium hydroxide in vacuo, an oil ofvitreous appearance is obtained which is used in the following operation(30 g).

5. Boc-Asp(OBzl)-Ile-Met-NH-NH-Troc

The preceding 30 g of oil in solution in 250 ml of THF are treated with6.4 ml (50 mM) of NEM and 18.9 (45 mM) of Boc-Asp(OBzl)O Su. Theapparent pH is adjusted between 6 and 7 by successive additions of NEM.After 4 hours, the product is isolated by evaporation of the THF,take-up in 400 ml of ethyl acetate followed by the same washings as thepreceding homologue (3). The residue after drying and evaporation (36.5g) is purified by chromatography over a column of silica withdichloromethane as solvent containing from 0 to 1.5% of methanol. 25 g(66%) of product presenting an NMR spectrum in accordance with theexpected structure are thus obtained.

m.p.=96°-100°.

6. Boc-Asp(OBzl)-Ile-Met-NH-NH₂

7.57 g (10 mM of Boc-Asp(OBzl)-Ile-Met-NH-NH-Troc in solution in 80 mlof the 80/20 mixture of DMF-AcOH are treated at ambient temperature with6.54 g of zinc in fine powder form with stirring for 30 mins. Theproduct is isolated by filtration of the zinc, treatment of the filtrateby 800 g of ice, then filtration after melting of the ice followed byrepeated washings with water. The white solid is firsly dried in airthen in a high vacuum in the presence of potassium hydroxide andphosphoric anhydride. 5.60 g (96%) of product with about 95% purityaccording to TLC and NMR are thus obtained. m.p.=180°-185° C.--Monitoredby TLC and HPLC.

EXAMPLE VI Boc-Arg-Lys(Z)-Leu-Leu-OH (fragment E₁) 1. Boc-Leu-Leu-OMe

In 500 ml of dioxane containing 18.4 g of NEM (0.16M), 9.05 g of H LeuOMe, HCl (0.05M), 7.4 g of HOBt (0.055M) and 18 g of Boc-Leu-ONSu(0.055M) are added successively. The product is stirred at ambienttemperature for 18 hours and the pH is adjusted to 6-7 by NEM, with pHpaper, if necessary in the course of reaction. The solvent isevaporated, the residue is dissolved in AcOEt and the organic solutionis successively washed twice with 5% KHSO₄ -K₂ SO₄, ClNa/water, 5%NaHCO₃, ClNa/water. The product is dried over MgSO₄ and the solvent isevaporated. The residue is dissolved in the minumum of ether and theproduct is precipitated by pentane, drained, dried.

Yield: 15 g (83%), TLC: chloroform-methanol-acetic acid, 95-5-3.

AcOEt-hexane 25/75--m.p.: 131-136

Possibility of purification over silica gel 60 Merck (70-230 mesh) inchloroform, with elution by the same solvent

2. H-Leu-Leu-OMe, TFA

27.5 g of Boc-Leu-Leu-OMe (0.076M) are covered with 180 ml of TFA atambient temperature: a slight heating occurs. After 30 mins. the solventis evaporated, the residual oil is taken up in ether and the product isscratched: a white precipitate is formed; pentane is added, the productis drained, dried.

Yield: 28.2 g (100%), TLC: chloroform-methanol-acetic acid 95/5/3,chloroform-methanol-acetic acid 80/15/5.

3. Boc-Lys(Z)-Leu-Leu-OMe

In 600 ml of AcOEt cooled with an ice bath, the following aresuccessively added:

28.2 g of H-Leu-Leu-OMe, TFA (0.076M)

26.22 g of NEM (0.0228M)

11.23 g of HOBt (0.083M)

46.4 g of Boc-Lys(Z)OTcp (0.083M).

The product is stirred, then the cold bath is removed. The pH isadjusted to 6-7 by NEM if necessary. After 18 hours, the organicsolution is successively washed with 5% KHSO₄ -K₂ SO₄, ClNa/water, 5%NaHCO₃, ClNa/water. The product is dried over MgSO₄. The solvent isevaporated, the residual oil is dissolved in chloroform and deposited atthe top of a column (L: 90 cm--diam. 5 cm) of silica gel 60 Merck(70-230 mesh) in chloroform. The product is eluted with chloroform,fractionated. The fractions containing the pure product are evaporated,the foam obtained is redissolved in ether and evaporated to dryness. Theresidue is triturated in pentane, drained and dried.

Yield: 42.4 g (91%), TLC: AcOEt/hexane 1/2, chloroform-MeOH-AcOH 95/5/3,Monitored by NMR-HPLC-EPP: 98.7%.

4. Boc-Arg-Lys(Z)-Leu-Leu-OMe

15.5 g of Boc-Lys(Z)-Leu-Leu-OMe (0.025M) are covered with 100 ml of TFAat ambient temperature. After 30 mins. with occasional stirring, thesolvent is evaporated completely. The residual oil is dissolved in 150ml of dioxane and the pH is brought to 6-7 by DIPEA, with pH paper. Theproduct is cooled with an ice bath and 7.21 g of Boc-Arg-OH, H₂ O, HCl(0.025M), 13.44 g of BOP (0.03M) and 10.32 g of DIPEA (0.08M) are added.The product is stirred, then the cold bath is removed and the pH isadjusted to 6-7 by DIPEA if necessary. After 18 hours, the dioxane isevaporated, the residual oil is dissolved in AcOEt and washedsuccessively with 5% NaHCO₃, ClNa/water. The product is dried overMgSO₄, the solvent is evaporated and the oil is dissolved in 5-95MeOH-chloroform, saturated with water, then deposited at the top of acolumn (L: 85 cm--diam. 4 cm) of silica gel 60 Merck (70-230 mesh) inthe same mixture. The product is eluted with:

1.5 l of MeOH-chloroform 5-95 saturated with water

1.1 of MeOH-chloroform 7.5-92.5 saturated with water

2.5 l of MeOH-chloroform

The product is fractionated and the pure fractions are evaporated, takenup in ether, evaporated and extracted completely in vacuo.

Yield: 16.8 g (87%), TLC: chloroform-MeOH-AcOH 95-5-3,chloroform-MeOH-AcOH 80-15-5, Monitored by NMR-HPLC-EPP 91.8%.

5. Boc-Arg-Lys(Z)-Leu-Leu-OH

13.98 g of Boc-Arg-Lys(Z)-Leu-Leu-OMe (0.018M) are solubilized in 250 mlof MeOH; 40 ml of water are added, then, at ambient temperature, 5.67 gof baryta (0.018M), finely pulverized. The product is stirred for 2 hrs.30 at ambient temperature. The end of reaction is monitored by TLC. CO₂is bubbled in up to pH 6, the product is filtered. The filtrate isevaporated, the residue is solubilized in isopropylic alcohol andfiltered, the alcohol is concentrated and the product is precipitated byether.

Yield: 12.31 (89%), TLC: chloroform-MeOH-AcOH 90/15/5,2-butanol-AcOH-water 72/7/21, Monitored by NMR-HPLC-EPP 95.88.

EXAMPLE VII Boc-Gln-Leu-Ser-Ala-OMe (fragment F₁) 1. Boc-Ser-Ala-Ome

25 g of H-Ala-OMe, HCl and 66 g. of Boc-Ser-ONb are dissolved in 500 mlDMF. The product is cooled in an ice bath and 25 ml of NEM, 24 g ofN-hydroxy benzotriazole are added with magnetic stirring and the pH ismaintained at 6.5 or 7 by addition of NEM. The product is stirred for 18hrs. at ambient temperature. After TLC monitoring, the medium isevaporated to 90% (0.1 mm Hg--35° C.). The oil obtained is dissolved in1.500 ml of chloroform, the product is washed with a solution ofsaturated sodium chloride (twice), 5% sulfate/potassium bisulfatesolution (3 times), saturated sodium bicarbonate (5 times). The solutionis dried over Na₂ SO₄ and evaporated to dryness. An oil is obtainedwhich is dried to constant weight (30°--0.1 mm Hg).

Yield: 53.78 g--Monitored by TLC.

2. H-Ser-Ala OMe, TFA

52.8 g of Boc-Ser-Ala-OMe are dissolved in 100 ml of methylene chloridewith magnetic stirring, being cooled with an ice bath. 250 ml of cooledTFA are added and the product is stirred for 20 mins. at ambienttemperature and filtered. The filtrate is evaporated to dryness (35°water-jet pump). By addition of 2 l of ether, a white solidprecipitates. It is drained, washed with ether (3 times), dried toconstant weight (30°--0.1 mm Hg).

Yield: 40.82 g.

3. Boc-Leu-Ser-Ala-OMe

40 g of H-Ser-Ala-OMe, TFA and 32.9 g of Boc-Leu-OH, H₂ O are dissolvedin 400 ml of DMF. The product is cooled with an ice bath with magneticstirring and 14.4 ml of NEM then 65.8 g of BOP and enough NEM tomaintain the pH at 6.5-7 are added. The product is stirred for 2 hrs. atambient temperature. After monitoring by TLC, the product is evaporatedto dryness (0.1 mm--35°) and the oil obtained is dissolved in 1500 ml ofchloroform, washed twice with a saturated NaCl solution, three timeswith 5% sulfate/potassium bisulfate, 3 times with saturated sodiumbicarbonate, dried over Na₂ SO₄, evaporated to dryness (30°--water-jetpump). The oil obtained is solidified in ether: Fraction A=29 g. Asecond jet B is obtained by addition of hexane, identical in TLC: B=13.7g.

Yield: 42.7 g. Monitored by NMR and TLC.

4. H-Leu-Ser-Ala-OMe, TFA

29 g of Boc-Leu-Ser-Ala-OMe are suspended in 50 ml of methylene chloridewith magnetic stirring, being cooled by an ice bath. 100 ml of cooledTFA are added and, after dissolution, the product is stirred for 30mins. at ambient temperature and filtered. The filtrate is evaporated todryness (35°--water-jet pump). The oil obtained is solidified in ether(the solvent is changed several times). The product is dried to constantweight (30°--0.1 mm Hg)

Yield: 30.2 g.

5. Boc-Gln-Leu-Ser-Ala-OMe

30 g of H-Leu-Ser-Ala-OMe, TFA and 25.5 g of Boc-Gln-ONp are dissolved,with magnetic stirring, in 300 ml of DMF. The product is neutralizedwith 9.7 ml of NEM. 10 g of OHBt are added and the product is stirredfor 2 hours at ambient temperature, maintaining the pH at 6.5-7 byaddition of NEM.

After monitoring by TLC, the medium is concentrated to 90% (0.1 mmHg--35°), taken up in 1000 ml of chloroform: a product precipitates intogel. The product is drained, washed, being taken up in 500 ml ofchloroform (twice) then in ether (3 times). The product is dried toconstant weight (0.1 mm Hg--30°).

Yield: 32.1 g, TLC: chloroform/MeOH/AcOH: 9/2/0.5--Rf=0.81 HPLC: EPP=91%AAA: Ser: 0.86-Glu: 1.02-Ala: 0.99-Leu: 0.99.

EXAMPLE VIII Boc-Tyr-Arg-Lys(Z)-Val-Leu-Gly-OH, HCl (fragment G₁) 1.Z-Leu-Gly-OCH₃

0.22 mole of Z-Leu-OH are dissolved in 500 ml of DMF. To this solutionare successively added:

25.11 g of HCl H-Gly-OCH₃ (0.2 mole)

28 ml of NEM

96 g of BOP (0.22 mole)

then the pH is adjusted to 7 by addition of NEM.

The reaction mixture is stirred at ambient temperature, maintaining thepH at 7 by addition of NEM if necessary. The reaction is followed in TLC(chloroform-methanol-acetic acid 95-5-9). After 2 hours, the reaction iscompleted. The reaction mixture is evaporated to dryness under reducedpressure (0.2 mm of mercury) and at a temperature lower than 30° C. Theresidue is taken up in ethyl acetate (1 liter).

The solution is washed successively with:

aqueous solution of bicarbonate (4×200 ml)

aqueous solution of SO₄ HK-SO₄ K₂ (4×200 ml)

saturated aqueous solution of sodium chloride (2×200 ml)

dried over magnesium sulfate and evaporated to dryness under reducedpressure. The residue is taken up in ether (500 ml), triturated,drained, dried.

Yield: 56.7 g (84.28%), TLC: chloroform-methanol-acetic acid 95-5-3 Rf:0.44.

2. HCl-H-Leu-Gly-OCH₃

56 g (0.166 mole) of Z-Leu-Gly-OCH₃ are dissolved in 900 ml of 0.23Nhydrochloric methanol. To this solution are added 6 g of 10% Pd/Ccontaining 50% humidity. Hydrogenation is carried out for 24 hours atambient temperature and under atmospheric pressure. The catalyst isfiltered and the solution is evaporated to dryness under reducedpressure at a temperature lower than 30° C. The residue is taken up inether (2×50 ml),. is decanted and the product is dried in a desiccatorin the presence of phosphoric anhydride. A white powder is obtained.

Yield: 33.53 g (84.4%), TLC: chloroform-methanol-acetic acid 90-20-9 Rf:0.15, Monitored by NMR.

3. Boc-Val-Leu-Gly-OCH₃

28.24 g of Boc-Val-OH (0.13 mole) are dissolved in 400 ml oftetrahydrofuran cooled to -10° C. 14.45 ml (0.13 mole) of isobutylchloroformate are then added. The mixture is stirred strongly at -10° C.for 15 minutes. To this mixture is added a solution of tetrahydrofurancooled to -10° C. containing 31.5 g of HCl H-Leu-Gly-OCH₃ (0.13 mole)previously neutralized by 14.45 ml of N methylmorpholine. Stirring iscontinued whilst cooling the reaction mixture in an ice bath (2 hours)then at ambient temperature for 2 hours. The reaction mixture isevaporated to dryness under reduced pressure at a temperature lower than30° C. The residue is taken up in a 500 ml-500 ml mixture of ethylacetate-water. The aqueous phase is decanted and the organic phasewashed successively with

an aqueous solution of sodium bicarbonate (2×50 ml)

an aqueous solution of SO₄ HK-SO₄ K₂ (pH 2) (2×50 ml)

an aqueous solution of sodium chloride (50 ml)

then dried over magnesium sulfate and evaporated to dryness underreduced pressure at a temperature lower than 30° C. The residue is takenup with pentane (50 ml), drained and dried in vacuo (0.1 mm of mercury).

Yield: 39.7 g (76%); TLC: chloroform-acetone 75-25 Rf: 0.46, Monitoredby NMR.

4. TFA H-Val-Leu-Gly-OCH₃

39 g of Boc-Val-Leu-Gly-OCH₃ (0.097 mole) are dissolved in 190 ml of TFAcooled in an ice bath. After dissolution, the ice bath is removed andthe mixture is stirred for 30 mins. at ambient temperature. The productis evaporated to dryness under reduced pressure and the residue is takenup in ether (100 ml), triturated and the ether is decanted. Thisoperation is repeated twice and the last traces of solvents areeliminated under reduced pressure (0.1 mm of mercury).

Yield: 44.8 g, TLC: chloroform-methanol-acetic acid 90-20-3 Rf: 0.36.

5. Boc-Lys(Z)-Val-Leu-Gly-OCH₃

25.55 g of TFA H-Val-Leu-Gly-OCH₃ (0.0615 mole) are dissolved in 200 mlof DMF. The solution is taken to pH 7 by addition of NEM. There are thenadded to this solution 32.47 g of Boc-Lys(Z)-OTCP (0.058 mole) and 9 gof HOBT (0.059 mole). The pH of the reaction mixture is returned to 7 byaddition of NEM. The product is stirred at ambient temperature,maintaining the pH at 7 by addition of NEM if necessary. The reaction isfollowed in TLC: chloroform-methanol, 90-10; chloroform-methanol-aceticacid, 90-20-3 (2 media).

After one hour, 1.96 g of Boc-Lys(Z)-OTCP (0.0035 mole) and 0.61 g ofHOBT are added. The pH of the solution is returned to 7 by addition ofNEM.

Two hours afterwards, the reaction is completed in TLC. The reactionmixture is then evaporated to dryness under reduced pressure (0.1 mm ofmercury) at a temperature lower than 30° C. The residue is taken up with500 ml of water, triturated. The solid formed is drained then washedwith

500 ml of aqueous solution of HKSO₄ -K₂ SO₄ (pH 2)

500 ml of a saturated aqueous solution of sodium bicarbonate

200 ml of water

then with ether (200 ml×2) and dried.

The impure product is chromatographed over a column of silica gel (5cm×150 cm). It is eluted with the 80-20 mixture of chloroform-ethylacetate (rate 600 ml/hr.)

The good fractions determined in TLC are collected together, evaporatedto dryness under reduced pressure. The residue is taken up in ether,triturated and the solid obtained drained. White powder, 35.14 g (86%).

TLC: chloroform-methanol 90-10 Rf: 0.62, chloroform-methanol-aceticacid, 87.7-9.4-2.8 Rf: 0.52, Monitored by NMR, AAA: AC Gly 1.00-Val0.96-Leu 1.06-Lys 0.98.

6. TFA H-Lys(Z)-Val-Leu-Gly-OCH₃

15 g of Boc-Lys(Z)-Val-Leu-Gly-OCH₃ (22.6 mM) are dissolved in a mixtureof TFA-methylene chloride (75 ml/75 ml). The product is stirred for 40mins. at ambient temperature, then evaporated to dryness under reducedpressure at a temperature lower than 30° C. The residue is taken up inether containing 20% of hexane (100 ml). The solid formed is drained anddried. A white solid of 14.51 g is obtained. Yield: 94.7%, TLC:chloroform/methanol/acetic acid (95/5/3) Rf: 0.12.

7. Boc-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OCH₃

14.1 g of TFA H-Lys(Z)-Val-Leu-Gly-OCH₃ (21 mM) are dissolved in 50 mlof DMF. The solution is taken to pH 6-7 by addition of NEM. 6.57 g ofBoc-Arg(HCl)-OH.1 H₂ O (20 mM) and 10.6 g of BOP (24 mM) are then addedto this solution. The pH of the reaction medium is returned to 6-7 byaddition of NEM. The product is stirred at ambient temperature,maintaining the pH at 6-7 by addition of NEM if necessary. The reactionis followed in TLC: chloroform/methanol (80/20).

After 5 hrs., the reaction is completed. The reaction mixture isevaporated to dryness under reduced pressure (0.1 mm of mercury) and ata temperature lower than 30° C. The oil obtained is taken up in 300 mlof ethyl acetate. The solid formed is drained and washed with ether (1stjet). Ether is added to the preceding filtrate; a solid precipitateswhich is drained (2nd jet): The two jets are identical in TLC. They arewashed in the solid state by stirring for 1 hour with a mixture of 200ml of ethyl acetate saturated water and 60 ml of water. The solid isdrained, then washed with 60 ml of water and 300 ml of ether and dried.

Yield: 78.7% (13.49 g), TLC: chloroform/methanol (20/20)--Rf=0.32,Monitored by NMR.

8. TFA H-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OCH₃

13.3 g of Boc-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OCH₃ (15.5 mM) are dissolvedin a (60 ml/60 ml) mixture of TFA-methylene chloride. The product isstirred for 40 mins. at ambient temperature then evaporated to drynessunder reduced pressure at a temperature lower than 30° C. The residue istaken up by a (80/20:100 ml) mixture of ether-hexane. The solid formedis drained, washed with hexane and dried (weight: 15.13 g).

TLC: chloroform/methanol (3/1)--Rf: 0.35

9. Boc-Tyr-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OCH₃

15.5 mM of TFA H-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OCH₃ are dissolved in 50 mlof DMF. The solution is taken to pH 6 by addition of NEM. 8.20 g ofBoc-Tyr-OTCP (15.5×10 mM) and 2.41 g of HOBT (15.5×10 mM) are then addedto this solution. The pH is returned to 6 by addition of NEM. Thereaction mixture is stirred at ambient temperature, maintaining the pHat 6 by addition of NEM if necessary. The reaction is followed in TLC:chloroform/methanol (3/1). After 3 hrs., the reaction is terminated. Thereaction mixture is evaporated to dryness under reduced pressure (0.1 mmof mercury) and at a temperature lower than 30° C. The residual oil istaken up in ethyl acetate (300 ml). The gelatinous solid formed isdrained, washed with ethyl acetate (100 ml), with ethyl acetate-ether(1/1:100 ml) then with ether (100 ml), finally with hexane (100 ml). Theproduct is dried under reduced pressure up to constant weight. Yield:96% (15.14 g).

TLC: chloroform/methanol (3/1)--Rf: 0.47--Monitored by NMR, AAA: AC:Gly: 0.95-Val: 0.98-Leu: 1.02-Lys: 1.01-Arg: 1.01--Tyr: 1.05, HPLC:93.81% EPP.

10. Boc-Tyr-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OH

7 g of Boc-Tyr-Arg(HCl)-Lys(Z)-Val-Leu-Gly-OCH₃ (6.86 mM) are dissolvedin a mixture of 70 ml of dioxane and 35 ml of water. 6.4 ml of 4N sodiumhydroxide (25.6 mM): 3.7 equiv.) are added to this solution and theproduct is stirred for 30 mins. at ambient temperature. It is dilutedwith 200 ml of water and 800 ml of ethyl acetate. The mixture is thenacidified to pH 3 by addition of (N) HCl. The ethyl acetate is decantedand the solid is drained (1st jet). The ethyl acetate is evaporated todryness; a 2nd jet is obtained. The two jets, identical in TLC, arecollected together, washed with ether and dried.

Yield: 74.7% (5.16 g), TLC: chloroform/methanol (2/1)--Rf: 0.45, 1Hmonitoring by NMR, AAA: Ac: Gly: 1.00-Val: 0.97-Leu: 1.04-Tyr: 0.95-Lys:0.99-Arg: 1.05 HPLC-EPP 91.52%.

EXAMPLE IX Boc-Ile-Phe-Thr-Asn-Ser-NH-NH₂ (fragment H₁) 1.Boc-Asn-Ser-OMe

A solution of 23.32 g of Boc-Asn-OH (0.1M) and 19.7 g of NbOH (0.11M) in250 ml of DMF, cooled on an ice bath, has 22.69 g of DCCI added thereto.After 3 hrs. reaction at ambient temperature, the DCU is filtered,cooled on an ice bath and 15.56 g of HCl, H-Ser-OMe are added. The pH istaken to, then maintained at 7 by addition of NEM. After 4 hrs. atambient temperature and 20 hrs. at +4° C., the solution is concentratedunder reduced pressure. The residue is taken up in the (50/50) mixtureof AcOEt/n-butanol. The organic solution is washed successively with:

a saturated solution of sodium bicarbonate

a saturated solution of sodium chloride

a solution of 5% KHSO₄ /K₂ SO₄

a saturated solution of sodium chloride.

After drying over MgSO₄ and evaporation under reduced pressure, theresidue is crystallized in the ether-hexane mixture. The product isfiltered, washed with ether/hexane and dried in vacuo yielding 26.9 g ofproduct.

Yield: 80.8%--Monitored by NMR and TLC

2. TFA, H-Asn-Ser-OMe

A solution cooled on an ice bath of 13.5 g of Boc-Asn-Ser-OMe (40.5 mM)in 54 ml of dichloromethane has 81 ml of TFA added thereto. After onehour of reaction at ambient temperature, the solution is concentratedunder reduced pressure, the residue is taken up with ether. A thick oilis obtained which is used in the following reaction.

3. Boc-Thr-Asn-Ser-OMe

A solution cooled on an ice bath of the crude product previouslyobtained in 300 ml of DMF has 14.09 g of Boc-Thr-ONSu (44.44 mM), 5.58 gof HOBt (41.3 mM) and NEM added thereto until a pH of 7 is obtained.After 20 hrs. of reaction at ambient temperature, the solution isconcentrated under reduced pressure and the residue is taken up in theAcOEt/n butanol mixture. The organic solution is successively washedwith:

a saturated solution of sodium bicarbonate

a saturated solution of sodium chloride

a solution of 5% KHSO₄ /K₂ SO₄

a saturated solution of sodium chloride.

After drying over MgSO₄, the solution is concentrated under reducedpressure and the residue crystallized in the ether/hexane mixture. 8.28g of product are obtained.

Yield: 47%--Monitored by NMR and TLC

4. TFA, H-Thr-Asn-Ser-OMe

A solution cooled on an ice bath of 8 g of Boc-Thr-Asn-Ser-OMe in 32 mlof dichloromethane has 54 ml of TFA added thereo. After one hour ofreaction at ambient temperature, the solution is concentrated underreduced pressure and the residue precipitated with ether. Afterfiltration, it is used as such in the following reaction.

5. Boc-Phe-Thr-Asn-Ser-OMe

A solution of the precipitate obtained in the preceding step in 100 mlof DMF has 7.82 g of Boc-Phe-ONp (20.24 mM), 2.53 g of HOBt (18.71 mM)and N-ethyl morpholine added thereto, with cooling by an ice bath, untila pH of 7 is obtained. After 4 hrs., the solution is concentrated underreduced pressure and the residue is taken up in AcOEt. The organicsolution is washed with:

a saturated solution of sodium bicarbonate

a saturated solution of sodium chloride

a solution of 5% KHSO₄ /K₂ SO₄

a saturated solution of sodium chloride

After drying over MgSO₄, concentration of the organic phase, the residueis crystallized in ether. 10 g of product are obtained.

Yield: 93.5% Monitored by TLC and NMR.

6. TFA, H-Phe-Thr-Asn-Ser-OMe

A solution of 10 g of Boc-Phe-Thr-Asn-Ser-OMe (17.19 mM) in 40 ml ofdichloromethane has 60 ml of TFA added thereto, with cooling by an icebath. After one hour of reaction at ambient temperature, the product isprecipitated with ether. The gum obtained after drying yields 5.14 g ofproduct.

Yield: 48.5%.

7. Boc-Ile-Phe-Thr-Asn-Ser-OMe

A solution cooled on an ice bath of 5.14 g of TFA, H-Phe-Thr-Asn-Ser-Ome(8.78 mM) in 100 ml of DMF has 3.17 g of Boc-Ile-OSu (9.66 mM), 1.30 gof HOBt (9.62 mM) and N-ethyl morpholine added thereto until a pH of 7is obtained. After 20 hours of reaction at ambient temperature, thesolution is concentrated under reduced pressure and the residue taken upin ether gives a precipitate which is filtered and dried in vacuo. 5.24g of product are obtained.

Yield: 85.9%--Monitored by NMR and HPLC.

8. Boc-Ile-Phe-Thr-Asn-Ser-NH-NH₂

A solution of 5 g of Boc-Ile-Phe-Thr-Asn-Ser-OMe (7.2 mM) in 40 ml ofDMF and 200 ml of methanol cooled on an ice bath has 3.6 ml of an 80%solution of hydrazine hydrate added thereto. After 21 hours of reactionat ambient temperature, 0.9 ml of the same solution of hydrazine hydrateare added. After 3 more hours, the gel obtained is filtered, washed withmethanol and dried in vacuo. 3.94 g of product are obtained.

Yield: 78.8%--Monitored by NMR and TLC.

EXAMPLE X Boc-Tyr-Ala-Asp(OBzl)-Ala-OH (fragment I) 1.Boc-Asp(OBzl)-Ala-OBut

3.62 g (0.02 mole) of hydrochloride of tertiobutyl alaninate aresuspended in 30 ml of acetonitrile. 2.52 ml of NEM are added then 8.4 g(0.02 mole) of Boc-Asp(OBzl) ONsu. The pH is maintained at 6-6.5 byaddition of NEM. Stirring is continued for 18 hours. After monitoring byTLC, the medium is evaporated to dryness, the oil is taken up in 50 mlof ACOET, the solution washed twice with a solution of KHSO₄ /K₂ SO₄,then with salted water, dried over Na₂ SO₄ and evaporated to dryness.The oil obtained is dissolved in hexane, a little solid is filtered andthe filtrate is evaporated to dryness. The oil obtained is used as such.

TLC: CHCl₃ /acetone/ACOH 80/15/5--Rf=0.8.

2. Boc-Ala-Asp(OBzl)-Ala-OH

The crude oil obtained previously is dissolved in 50 ml of iced TFA andstirred for 1 hr. 30 mins. at ambient temperature. The TFA is evaporatedto a maximum and the residual oil is taken up three times in anhydrousether and decanted. It is then dried in a desiccator in vacuo in thepresence of pentane. A solid foam is formed which is crushed andreturned to be dried.

9.4 g of crude product is obtained, used as such for coupling.

The above TFA salt is dissolved in a mixture of 20 ml of DMF and 18 mlof distilled water. NEM is added up to a pH of 7 (using the pH-meter). 4g (0.014 mole) of Boc-Ala-ONsu in solution in 16 ml of DMF areintroduced. The pH is maintained at 6.5-7 by addition of NEM. After 5hrs. 30 mins., the reaction is terminated (monitored by TLC). Thesolvent is evaporated to a maximum and the residue is taken up in amixture of water and ethyl acetate. The organic phase is washed with asolution of NaHCO₃, with salted water, dried over Na₂ SO₄ and evaporatedto dryness. The residue is dissolved in ether and precipitated by hexaneto give a gum which crystallizes after trituration.

The solid is drained and dried in air.

Yield: 5.05 g TLC: CHCl₃ -acetone-ACOH 80-15-5--Rf=0.28.

3. Z-Tyr-Ala-Asp(OBzl)-Ala-OH

1 g (2 mmoles) of Boc-Ala-Asp(OBzl)-Ala-OH is introduced into 10 ml ofstirred ice TFA.

After 30 mins. of stirring at ambient temperature, the TFA is evaporatedto a maximum, the residue taken up twice in anhydrous ether anddecanted, then dried in a desiccator in vacuo in the presence ofpotassium hydroxide. A solid foam is formed.

The crude product is dissolved in a mixture of 4 ml DMF and 2 ml ofdistilled water. NEM is added up to pH 7-7.5. 1 g (2.4 mmoles) of1-Tyr-ONsu is then added and the pH is maintained at 7-7.5 by NEM(pH-meter). After 2 hrs. 30 mins., after monitoring by TLC, the solventis evaporated to a maximum at 40° C. The residue is taken up in ethylacetate and washed with water; a little solid is eliminated and theethyl acetate is washed with the mixture of sulfate bisulfate and saltedwater. After the product has been left for a few hours, a precipitate isformed which is drained, washed with ether and dried in air.

500 mg are thus obtained. A 2nd jet of 350 mg, slightly less pure, isobtained by concentration of the ethyl acetate.

TLC chloroform-ACOH 3-1 Rf: 0.4, butanol-ACOH-H₂ O 72-7-2 Rf: 0.85,chloroform-acetone-ACOH 80-15-5 Rf: 0.1.

HPLC 90.9% polypeptide purity.

Monitored by NMR.

EXAMPLE XI Synthesis of:H-Arg-Lys(Z)-Leu-Leu-Gln-Asp(Bzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂(peptide K) 1. Boc-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Leu-NH₂, 3 HCl

34.47 g of H-Ala-Arg-Ala-Arg-Leu-NH₂, 3 HCl (57 mM) are dissolved in 230ml of DMF. 28 g of BOP (63 mM) are added, then in one hour 33.5 g (57mM) of Boc-Glu(OBzl)-Arg-Gly-OH in solution in 190 ml of DMF. The pH ofthe reaction medium is maintained at around 6 by addition of NEM. After4 hours, the reaction medium is concentrated in vacuo by half and theresidue poured over 800 ml of ethyl acetate. The precipitate obtained isfiltered, washed with ethyl acetate, with ether and dried in vacuo. 56.8g of product are obtained with a yield of 80%.

Identification: 1H NMR and AAA: Glu: 0.98-Gly: 1.01-Ala: 1.97-Leu:1.05-Arg: 2.97.

HPLC reverse phase EPP 95%--TLC in EPAW 40/20/10/6--Rf=0.55.

2. H-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂, TFA, HCl

55.3 g of the above product are stirred for 30 mins. in 250 ml of CH₂Cl₂ +250 ml of TFA. Then the reaction medium is concentrated in vacuo byhalf and poured over 1200 ml of iced ether. The precipitate obtained isfiltered, washed with ether, dried in vacuo over potassium hydroxide.Yield: 100%.

3.Boc-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl

To the preceding TFA salt (45 mM) dissolved in 400 ml of DMF andneutralized by NEM, are added 22.2 g of BOP (50 mM) then, in one hour,30 g (45 mM) of Boc-Glu(OBzl)-Ser-Asn-Gln-OH in solution in 300 ml ofDMF. The pH of the reaction medium is maintained at 6. After 6 hours,the medium is concentrated by half in vacuo at 30° C. then poured over1200 ml of ethyl acetate. The precipitate formed is filtered, washedwith ethyl acetate, then with ether and finally dried in vacuo.

Yield: 60.5 g (75%).

Product identified by NMR and AAA: Asp: 1.00-Ser: 0.81-Glu: 2.71-Gly:0.89-Ala: 1.82-Leu: 0.96-Arg: 3.02-HPLC reverse phase EPP 70%--TLC: EPAW30/20/10/6: Rf: 0.35.

4. H-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,TFA, HCl

53.3 g (30 mM) of the above product are stirred for 35 mins. in 250 mlof dichloromethane plus 250 ml of TFA. After concentration in vacuo byhalf, the residue is poured over 1300 ml of iced ether; the precipitateformed is filtered, washed with ether, then dried in vacuo overpotassium hydroxide.

5.Boc-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl

The preceding TFA salt is dissolved in 300 ml of DMF and neutralized byNEM. 14.8 g of BOP (33 mM) are added then, in small portions in onehour, 21.4 g (30 mM) of Boc-Ser-Arg-Gln-Gln-Gly-OH. The pH of thereaction medium is maintained at around 6 by NEM. After 7 hours, themedium is poured over 2 l of ACOEt. The precipitate obtained isfiltered, washed with ethyl acetate, then with ether and finally driedin vacuo. Yield: 57 g (80%).

Product identified by NMR, AAA-Ser: 1.7-Arg: 4.1-Glu: 4.7-Gly: 2.05-Asp:1.00-Ala: 1.91-Leu: 0.97-TLC BPEW1 Rf: 0.35.

6.H-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,TFA, HCl

14.21 g (6 mM) of the preceding product in suspension in 80 ml ofdichloromethane are treated in 10 mins. in an atmosphere of nitrogen by120 ml of trifluoroacetic acid. After 45 mins. at ambient temperaturewith stirring, the product is concentrated in vacuo to 1/3 of theinitial volume and is poured over 750 ml of stirred cold ether (+4° C.).The precipitate is filtered and washed with ether then dried in vacuoover potassium hydroxide then phosphoric anhydride. 13.9 g (97%) ofproduct are obtained, used as such in the following reaction.

7.Boc-Asp-(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl

5.82 g (10 mM) of Boc-Asp(OBzl)-Ile-Met-NH-NH₂ in solution in 76.6 ml ofDMF are treated between -20° and -25° with 6.66 ml (40 mM) of gaseous6.0N HCl/dioxane, then with 1.45 ml (12 mM) of tertiobutyl nitrite insolution in 5 ml of DMF. After one hour between -20° and -25°, theproduct is neutralized to an apparent pH of 6-7 by addition of about 8ml of diisopropylethylamine (DIPEA) and the total volume is adjusted to100 ml with a little DMF, which leads to a 0.1 molar solution of azide(which is always conserved at around -20°).

13.84 g (about 5.8 mM) of the preceding TFA salt dissolved in 58 ml ofDMSO and 87 ml of DMF are neutralized to an apparent pH of 6.7 with 1 mlof DIPEA and the solution thus obtained has 72.5 ml (or about 1.25equiv.) of the above azide solution added thereto in about 10 mins. at atemperature of between -20° and -25°. The pH is adjusted to 6-7 by alittle DIPEA and the solution conversed at -15°. After 16 hours, afurther 14.5 ml (0.25 equiv.) of the azide solution is added. Monitoringby TLC shows a complete reaction after 24 hours, but reaction is stoppedonly after 40 hours by pouring the solution over 750 ml of stirred iceethyl acetate.

The precipitate thus formed is filtered and washed abundantly with thesame solvent and dried in vacuo in the presence of phosphoric anhydride.14.35 g are thus obtained, used as such in the following step.

8.H-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,TFA, HCl

14 g of the above product are stirred for 35 minutes in 80 ml of CH₂ Cl₂+120 ml of TFA plus 10 ml HS(CH₂)₂ SH. After concentration in vacuo byhalf, the reaction medium is poured over 800 ml of iced ether. Theprecipitate obtained is filtered, washed with ether then dried in vacuoover potassium hydroxide.

Yield: 100%.

9.Boc-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl

To the TFA salt obtained above (4.9 mM) dissolved in 120 ml of DMF plus30 ml of DMSO and neutralized by NEM, are added 740 mg of HOBt (5.5 mM)then, in small portions, 2.02 g of Boc-Gln-ONp (5.5 mM), the pH of thereaction medium is maintained at around 6 by addition of NEM. After 6hours, the reaction medium is poured in 800 ml of AcOEt. The precipitateobtained is filtered, washed with ethyl acetate, then with ether, thendried in vacuo.

Weight: 14.8 g--Yield: 100%.

The product is identified by NMR and AAA: Asp: 1.97-Ser: 2.0-Glu:6.12-Gly: 2.06-Ala: 2.06-Met: 0.55-Ile: 0.93-Leu-0.99-Arg: 3.81-TLC: Rf0.45-BPEW1

10.H-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,TFA, HCl

14.7 of the above product are stirred for 35 mins. in 80 ml of CH₂ Cl₂plus 10 ml HS(CH₂)₂ SH+120 ml of TFA. After concentration in vacuo byhalf, the reaction medium is poured over 600 ml of iced ether. Theprecipitate obtained is filtered, washed with ether then dried in vacuoover potassium hydroxide.

Weight: 14.5 g.

11.Boc-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl

To the TFA salt obtained hereinabove (4.9 mM) dissolved in 120 ml of DMFplus 40 ml of DMSO and neutralized by NEM, 2.44 g of BOP (5.5 mM) and4.2 g of Boc-Arg-Lys(Z)-Leu-Leu-OH, HCl (5.5 mM) are added in smallportions in 30 mins. The pH of the reaction medium is maintained ataround 6 by addition of NEM. After 23 hrs., the reaction medium ispoured over one liter of AcOEt. The precipitate formed is filtered,washed with AcOEt then Et2O, then dried in vacuo.

M: 16.5 g--Yield: 93%--Product identified by NMR and AAA: Asp: 2.10-Ser:1.86-Glu: 6.2-Gly: 2.00-Ala: 2.00-Met: 0.55-Ile: 0.80-Leu: 2.89-Lys:0.87-Arg: 4.67-TLC Rf: 0.40 (BPEW1).

EXAMPLE XII Synthesis of:Boc-Ile-Phe-Asn-Thr-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-OH,HCl (peptide J) 1. H-Gln-Leu-Ser-Ala-OCH₃

10.3 g (18.8 mM) of Boc-Gln-Leu-Ser-Ala-OCH₃ are stirred for 35 mins. in100 ml of dichloromethane containing 100 ml of TFA. The reaction mediumis concentrated in vacuo to about 50 ml and poured over 300 ml of ether.The precipitate is drained, washed with ether and dried in vacuo.

2. Boc-Tyr-Arg(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-OCH₃, HCl

To the TFA salt obtained hereinabove, dissolved in 200 ml ofdimethylformamide and neutralized with N-ethylmorpholine are added 18.9g (18.8 mM) of Boc-Tyr-Arg-Lys(Z)-Val-Leu-Gly-OH then 9.2 g of BOP (21mM) then NEM to take the pH of the reaction medium to around 6. After 4hrs. of stirring, the reaction medium is poured over 1 liter of AcOEt.The precipitate obtained is filtered, washed three times with 100 ml ofAcOEt, then 3 times with 100 ml of Et2O then it is dried in vacuo.

24.16 g of product are obtained. Yield: 90% TLC Rf: 0.20 (BEW1)--alphaD: -17.6°--(Cc: 1-DMF with 5% AcOH)--Product identified by 1H NMR andAAA: Ser: 1.01-Glu: 0.99-Gly: 1.06-Ala: 1.01-Val: 0.98-Leu: 1.97-Tyr:1.00-Lys: 0.98-Arg: 0.99-HPLC in reverse phase (EPP: 92%).

3. H-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Arg-OCH₃, HCl, TFA

8.66 g (6.1 mM) of the above product are stirred for 35 mins. in 37 mlof dichloromethane plus 3.5 ml of anisole plus 40 ml of TFA. Afterconcentration in vacuo by half, the residue is poured over 300 ml oficed ether; the white precipitate obtained is filtered, washed withether then dried in vacuo over potassium hydroxide.

Weight: 8.68 g--Yield: 100%.

4. Boc-Ile-Phe-Thr-Asn-Ser-N₃

5.94 g (8.55 mM) of Boc-Ile-Phe-Thr-Asn-Ser-NH-NH₂ are dissolved in 27ml of DMSO plus 37 ml of DMF. After cooling to -25° C., 6.1 ml of a 5.6N HCl/dioxane solution then 1.23 ml of tertiobutyl nitrite (1.2 equiv.)prediluted in 5 ml of DMF precooled to -20° C., are added. After 1 hr.15 mins. of stirring between -20° and -25° C., 6.1 ml ofdiisopropylethylamine (DIPEA) are added to take the pH to around 6, then3 ml of DMF are added to take the volume to 85 ml and this solution isconserved at -25° C.

5.Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-OCH.sub.3,HCl

The TFA salt obtained in Example XI-3 is dissolved in 70 ml of DMF andneutralized by DIPEA then added in 15 mins. to 70 ml of the azidesolution obtained hereinabove at -20° C. Then DIPEA is added to take thepH towards 7, the product is stirred for one hour at -20° C., then themedium is conversed at -12° C. After 22 hrs., 5 ml of the azide solutionare added and the pH is raised towards 7 by DIPEA. After 45 hrs., theremaining azide solution is added and the pH is raised towards 7 byDIPEA. After 74 hrs., the reaction medium is poured over 1200 ml ofethyl acetate. The white precipitate obtained is filtered, washed withethyl acetate then with ether, then dried in vacuo.

Weight: 10.46 g--Yield: 86%.

By concentration of the mother liquors and reprecipitation by an ethylacetate/ether mixture, a second jet is obtained which makes it possibleto have an overall yield of 95%. The coupling product is identified by1H NMR and AAA: Asp: 1.02-Thr: 1.03-Ser: 1.91-Glu: 0.97-Gly: 1.06-Ala:1.06-Val: 1.00-Ile: 0.94-Leu: 1.98-Tyr: 1.04-Phe: 1.02-Lys: 0.94-Arg:1.02-TLC Rf: 0.20 (BEW1)--HPLC in reverse phase (EPP: 90%).

6.Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Gly-Gln-Leu-Gly-Gln-Leu-Ser-Ala-OH,HCl

5.3 g (2.7 mM) of the product obtained hereinabove are dissolved in 40ml of DMSO plus 6 ml of water; 10 ml of 1N sodium hydroxide are addeddrop by drop, the product is stirred for 30 mins. then neutralized with10 ml of 1N hydrochloric acid. The reaction medium is poured over oneliter of ethyl acetate. The precipitate obtained is filtered, washed 4times with ethyl acetate then twice with water, then 4 times with ether,then it is dried in vacuo over P₂ O₅.

Weight: 4.56 g--Yield: 86%--Product identified by 1H NMR and AAA--TLC:Rf: 0.5 BEW1.

EXAMPLE XIII Synthesis ofZ-Tyr-Ala-Asp(OBzl)-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl (GRF 1-44 protected) 1.H-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Glu-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl, TFA

1.4 g of the product of Example X-11 are stirred for 35 mins. in 60 mlof CH₂ Cl₂, plus 70 ml of TFA+7 ml of ethane dithiol. Afterconcentration in vacuo by half, the residue is poured over 500 ml of iceether. The precipitate obtained is filtered, washed with ether, thendried in vacuo over potassium hydroxide. (Weight: 7.44 g).

2.Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl

To 6.6 g of the above TFA salt (1.9 mM) dissolved in 36 ml of DMSO plus90 ml of DMF and neutralized by NEM, 1.0 g of BOP (2.3 mM) then 3.8 g ofthe peptide of Example XI-6 (1.9 mM) are added, the pH of the reactionmedium is taken to 6 with NEM. After 19 hours, 1.15 mM of BOP are added.After 26 hrs., the reaction medium is poured over 600 ml of AcOEt. Theprecipitate obtained is filtered, washed with AcOEt, then filtered anddried in vacuo. Yield: 88% (9.2 g)--product identified by NMR and AAA.

3.H-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂,HCl, TFA

9.1 g of the above product are stirred for 35 mins. in 100 ml of TFAplus 5 ml of ethane dithiol. The reaction medium is then poured over 500ml of iced ether. The precipitate obtained is filtered, washed withEt2O, then dried in vacuo over potassium hydroxide.

Yield: 100%.

4. Protected GRF-1-44

To 9.10 g of the preceding TFA salt (1.66 mM) in solution in 50 ml ofDMF and 50 ml of DMSO and neutralized by NEM, are added 1.18 g of thepeptide described in Example IX-3 (1.78 mM) and 0.79 g of BOP (1.78 mM);the pH is maintained at around 6 by NEM. After 23 hrs. at ambienttemperature, the reaction medium is poured over 600 ml of AcOEt. Theprecipitate formed is filtered, washed with ethyl acetate, then withether and finally dried in vacuo.

Yield: 8.54 g (84%), Monitored by NMR and TLC:-AAA: Asp: 4.2-Thr:0.92-Ser: 4.02-Glu: 7.37 (for 7)-Gly: 3.14 (for 3)-Ala: 5.11-Val:1.01-Met: 0.61-Ile: 1.84-Leu: 4.98-Tyr: 1.87-Phe: 0.95-Arg: 5.90.

EXAMPLE XIV Deprotection of GRF 1-44

4.5 g of the product obtained in accordance with Example XII-4 arestirred for one hour at 0° in 180 ml of TFA containing 19.8 ml of TFMSA,27 ml of thioanisole and 11 of metacresol. 1 l. of ether is then added.The precipitate obtained is filtered, washed with ether then dried onehour in vacuo in the presence of potassium hydroxide. The product isredissolved in 200 ml of water and ion exchanger resin Amberlite IR 45(in the form of acetate) is added in order to return the pH to 4.5. Themedium is stirred for 30 mins. at ambient temperature. The resin isfiltered, washed with water and the filtrates concentrated to 50 ml thenlyophilized. Yield: 3.85 g. Monitored by NMR of the disappearance of theprotector groups Z and OBzl.

TLC (BEPW1): majority spot Rf 0.38.

EXAMPLE XV Purification of the GRF 1-44

The deprotected peptide obtained in the preceding Example (XIII) issubjected to a chromatography over Sephadex G 50 gel (fine), using 30%acetic acid as eluent.

The fractions containing the expected peptide are collected together,evaporated and lyophilized.

The lyophilizate thus obtained is purified in turn by a chromatographyover a cation exchanger of the CM-32 carboxymethylcellulose (Whatman)type, using a linear gradient of ammonium acetate of between 01M (pH4.5) and 0.4M (pH 6.5). For example, for 1 charge of 1 g of peptides tobe purified, a chromatography column having a bed volume of about 50 mlfor a height of 20 cm will be used.

The fractions containing the peptide with a degree of purity ≧80% (HPLC)are collected together and lyophilized up to constant weight (in orderto eliminate the CH₃ CO₂ NH₄) buffer.

Finally, the preceding lyophilizate is subjected to a partitionchromatography, using as support of the stationary liquid phase, fineSephadex G 50 and with the aid of the following system of solvents:n-butanol/ethanol/pyridine/0.2N acetic acid in the proportion of 4/1/1/7(in volume). The fractions of chromatography are monitored by HPLC andthose of which the titer of purity is ≧95% are collected together andlyophilized.

The product is finally monitored by analysis of the amino acids:

Tyr: 1.91 (2)

Ala: 4.88 (5)

Aln, Asp: 3.86 (4)

Ile: 1.96 (2)

Phe: 0.93 (1)

Thr: 1.02 (1)

Ser: 3.88 (4)

Arg: 5.89 (6)

Lys: 1.98 (2)

Val: 1.01 (1)

Leu: 5.10 (5)

Gly: 2.98 (3)

Gln, Glu: 6.82 (7)

Met: 0.91 (1)

EXAMPLE XVI Synthesis of Boc-Glu-Ser-Asn-Gln-Glu-Arg-Gly (fragment B)1-Z-Arg(NO₂)-Gly-OBzl

353 g of tosylate of glycine benzyl ester (H-Gly-o-Bzl, Tosylate) aredissolved in 2 liters of DMF; 115 g of NEM then 353 g of Z-Arg(NO₂)-OHand finally 500 g of BOP are added. The pH of the medium is adjusted to7 with the aid of NEM and using pH indicating paper on samples of thereaction medium diluted with water. The reaction medium is stirred andthe state of advance of the reaction followed by TLC. After 4 hrs., thereaction is complete and the medium evaporated to dryness in vacuo at35°. The residue of evaporation is taken up in 2 liters of ethyl acetateand 2 liters of water. Under these conditions, a solid is obtained whichis drained and washed successively in solid phase with a 5% aqueoussolution of HKSO₄ +K₂ SO₄, with pure water, with a 5% aqueous solutionof NaHCO₃ and finally water.

The product is dried in air and monitored by NMR and TLC.

Yield: 410 g (82%).

2. H-Glu(OBzl)-Arg-Gly-OH

500 g of the preceding product in solution in 3 liters of HClN in DMFand in the presence of 100 g of Pd/C with 10% of Pd are hydrogenatedunder a pressure of 1.5 bar of hydrogen. After 24 hours, the reaction isterminated (monitored by TLC). The catalyst is filtered and the pH ofthe medium adjusted to 7 by addition of N-ethyl morpholine and using pHindicating paper on samples diluted with water. 458 g of Boc-Glu(OBzl)-ONp are introduced in solution in 1 liter of DMF. The reaction medium isdiluted by addition of 1 liter of water and 135 g of OHBT and NEM areprogressively added in sufficient quantities to maintain the pH at 7.

The medium is stirred for 4 hours at ambient temperature. One makes surethat the reaction is complete and the solvent is evaporated in vacuo atthe temperature of 35°. The residue of evaporation is taken up in ethylacetate. Under these conditions, the product becomes solid. It isdrained and chromatographed over silica gel, using chloroform-methanol(70-30 by volume) as eluent. Evaporation of the fractions ofchromatography containing the pure product leads to 412 g (75%) of awhite powdery compound after lyophilization. It is monitored by TLC andNMR.

The preceding product is dissolved at ambient temperature in 3 liters ofa (50/50) mixture of TFA and CH₂ Cl₂. The medium is maintained for 15mins. at ambient temperature, and is evaporated to dryness in vacuo,maintaining the temperature at 20°. The residue is taken up in ethylether and the solid obtained is drained and dried. The yield is 423 g(100%) in the form of trifluoroacetate.

3. H-Gln-Glu(OBzl)-Arg-Gly-OH

564 g of trifluoroacetate of H-Glu(OBzl)-Arg-Gly-OH are dissolved in 3liters of dimethyl formamide. The pH is adjusted to 7 by addition of NEMon samples diluted with water and with the aid of pH indicating paper.

367 g of Boc-Gln-ONp arethen introduced in solution in 1 liter of DMF.The reaction medium is diluted by addition of 1 liter of water. 135 g ofOHBT and the sufficient quantity of NEM for the pH to be maintained at 7are progressively added.

The mixture is stirred for 4 hours at ambient temperature. The end ofthe reaction is monitored by TLC and the solvent is evaporated in highvacuum at 35°. The residue of evaporation is triturated in ethylacetate. Under these conditions, a hygroscopic solid is obtained whichis used as such for the treatment with trifluoroacetic acid--CH₂ Cl₂ (2liters of the 50-50 by volume mixture).

The medium is conserved for 10 minutes at ambient temperature andfinally evaporated in vacuo at 20°. The residue of evaporation istriturated in ethyl ether to yield 525 g (76%) ofH-Gln-Glu(OBzl)-Arg-Gly-OH. (Monitored by NMR and TLC).

4. H-Asn-Gln-Glu(OBzl)-Arg-Gly-OH

By using the conditions of Example II-2 strictly and fromH-Gln-Glu(OBzl)-Arg-Gly-OH, trifluoroacetate (691 g) and 393 g ofBoc-Asn-ONb, 685 g (85%) of a solid white product are obtained,monitored by NMR and TLC.

5. H-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-OH

Likewise under the conditions of Example II-2 and from 806 g ofH-Asn-Gln-Glu(OBzl)-Arg-Gly-OH and 366 g of Boc-Ser-ONb, the product isobtained, monitored by NMR and TLC with a yield of 688 g (77%) and intrifluoroacetate form.

6. Boc-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-OH (fragment B)

From 894 g of H-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-OH trifluoroacetate and458 g of Boc-Glu(OBzl)-ONp are obtained 824 g (80%) of the expectedproduct after purification by crystallization in the DMF-ether (50-50 byvolume) medium.

The product is monitored by NMR, TLC and analysis of amino acids:

Gln-Glu: 2.92 (3)

Ser: 0.97 (1)

Asn: 1.03 (1)

Arg: 0.97 (1)

Gly: 0.96 (1)

By following the strategies presented in Tables V, VI, VII and VIII andthe techniques described in Examples I to IX and XVI, the followingcompounds may be obtained:

Boc-Lys(Z)-Leu-Leu-Gln-OH (fragment E)

Boc-Gln-Leu-Ser-Ala-Arg-OH (fragment F)

Boc-Lys(Z)-Val-Leu-Gly-OH (fragment G)

Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-OH (fragment H)

EXAMPLE XVIIBoc-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂(fragment B-A)

69.2 g of fragment A hydrochloride are dissolved in 500 ml of DMF. ThepH is adjusted to 7 on samples diluted with water and with the aid of pHindicating paper and NEM. 117 g of fragment B, 55 g of BOP are thenadded, the pH is again adjusted to 7 with the aid of NEM and the mediumis stirred for 14 hours at ambient temperature. After monitoring the endof reaction by TLC, 2 liters of ethyl ether are added to the reactionmedium. A white powdery precipitate is deposited under these conditions.It is purified by recrystallization in DMF-ethyl acetate. The drainedproduct is then washed with ethyl acetate in solid phase and dried.

149 g (81%) of a white product are obtained, monitored by TLC and NMR.Analysis of amino acids:

Gln-Glu: 2.92 (3)

Ser: 1.02 (1)

Asn: 0.97 (1)

Arg: 2.89 (3)

Gly: 1.01 (1)

Ala: 1.98 (2)

Leu: 0.93 (1)

DEPROTECTION OF N-TERMINAL NITROGEN

The product described in the preceding Example (184 g) is introduced ina mixture constituted by 750 ml of TFA and 750 ml of CH₂ Cl₂ withstirring and at ambient temperature. After dissolution, the mixture isconserved for 15 mins. at the same temperature and evaporated in vacuo,maintaining the medium of evaporation at 25°. The residue of evaporationis triturated in ether and, under these conditions, a white powderysolid is obtained, which is dried in vacuo and monitored by TLC and NMR.

The following compounds may be obtained in accordance with thetechniques of Example XVII and representing the different stages ofelongation of the peptide.

Fragment: C-B-A

Boc-Ser-Arg-Gln-gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu-(H)Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂ ##STR35##

In this particular case, fragment D is introduced, using the activationof the azides ##STR36##

As soon as the methionine is in sequence, a scavenger will be used inthe course of the different treatments by TFA-CH₂ Cl₂ during the phasesof selective deprotection (elimination of the Boc). The inventionrecommends thioanisole as agent for protecting the methionine fromoxidation in the proportion of 5% in the TFA-CH₂ Cl₂ medium.

Fragment: E-D-C-B-A

Boc-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln (H)Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH.sub.2

Fragment: F-E-D-C-B-A

Boc-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl) (H)Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂

Fragment: G-F-E-D-C-B-A

Boc-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Ser-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂

Fragment: H-G-F-E-D-C-B-A

Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-(H)-Gly-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂

protected hPGRF

Z-Tyr-Ala-Asp(OBzl)-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂

Deprotection and purification of the hpGRF-1-44 are then carried out asindicated in Examples XIV and XV.

What is claimed is:
 1. A process for the synthesis, in liquid phase andby fragments, of hpGRF 1-44 and hpGRF 1-40, comprising the stepsof:coupling, one after the other and in the order of the sequence of theGRF, the following fragments: H-Ala-Arg-ala-Arg-Leu-NH₂ (40-44), calledfragement A or alaninamide;Boc-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-OH (33-39), called fragmentB; Boc-Ser-Arg-Gln-Gln-Gly-OH (28-32), called fragment C;Boc-Asp(OBzl)-Ile-Met-NH-NH₂ (25-27), called fragment D;Boc-Lys(Z)-Leu-Leu-Gln-OH (21-24), called fragment E;Boc-Gln-Leu-Ser-Ala-Arg-OH (16-20), called fragment F;Boc-Lys(Z)-Val-Leu-Gly-OH (12-15), called fragment G;Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-OH (5-11), called fragment H; andZ-Tyr-Ala-Asp(OBzl)-Ala-OH (1-4), called fragment I;wherein (a) the sideacid functions of the aspartic and glutamic acids and the side aminefunction of the lysine are protected by protector groups stable underthe conditions of deprotection of the group Boc, (b) the guanidinefunction of the arginine is protected by protonation, and (c) theN-terminal amino acid is protected on the amine by the Boc group;selectively eliminating the group Boc from the N-terminal amine of thepeptide in phase of elongation by hydrolysis with trifluoroacetic acid,said coupling being effected in an aprotic polar solvent; andeliminating, at the end of the sequence, all the protector groups byhydrolysis with the aid of a 0.1 to 1M solution of methanesulfonic ortrifluoromethanesulfonic acid in trifluoroacetic acid.
 2. The process ofclaim 1, wherein, after each coupling, the product obtained is isolatedfrom the reaction medium by precipitation with the aid of an apolarsolvent.
 3. The process of claim 1, wherein the crude product ofreaction is purified by counter-current distribution and permeationchromatography over gel.
 4. The process of claim 1, wherein alaninamideis used in place of fragment A and the peptide obtained is hpGRF 1-40.5. The process of claim 1, wherein the following three peptide fragmentsare coupled successively and in the order of the sequence of the GRF:(1)H-Arg-Lys(Z)-Leu-Leu-Gln-Asp(OBzl)-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu(OBzl)-Ser-Asn-Gln-Glu(OBzl)-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂(sequence 20-44 of the GRF, called peptide K) (2)Boc-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys(Z)-Val-Leu-Gly-Gln-Leu-Ser-Ala-OH(sequence 5-9 of the GRF called peptide J); (3)Z-Tyr-Ala-Asp(OBzl)-Ala-OH (sequence 1-4 of the GRF called peptide I);said peptide obtained being hpGRF 1-44.
 6. The process of claim 5,wherein the peptide fragment corresponding to sequence 20-40 of the GRFis used in place of the peptide K, said peptide obtained being hpGRF1-40.
 7. The process of claim 1, wherein the coupling is carried outwith the aid of the hexafluorophosphate of benzotriazolyloxyphosphonium(BOP) or dicyclohexyl-carbodiimide in the presence of 1-hydroxybenzotriazole (coupling agent) or by activation with carboxyazides, indimethylformamide or dimethylsulfoxide.